HotStarTaq Master Mix Kit

For highly specific amplification for any PCR application

  • High PCR specificity without the need for optimization
  • Easy reaction setup at room temperature
  • Ready-to-use master mix format reduces pipetting steps
HotStarTaq Master Mix contains HotStarTaq DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization, and dNTPs. Providing all components in a master mix reduces pipetting steps and the risk of contamination, while increasing throughput and reproducibility.
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HotStarTaq Master Mix Kit (250 U)
3 x 0.85 ml HotStarTaq Master Mix (contains 250 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP)and 2 x 1.7 ml RNase-Free Water
 203443
£138.00
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HotStarTaq Master Mix Kit (1000 U)
12 x 0.85 ml HotStarTaq Master Mix (contains 1000 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 8 x 1.7 ml RNase-Free Water
 203445
£468.00
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Hot StarTaq Master Mix Kit (2500 U)
1 x 25 ml HotStarTaq Master Mix (contains 2500 units HotStarTaq DNA Polymerase PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 1 x 50 ml RNase-Free Water
 203446
£984.00
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The HotStarTaq Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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HotStarTaq procedure.|Superior performance.|Higher specificity with different primer–template systems.|Effect of hot start on RT-PCR performance.|Highly sensitive single-cell PCR.|Tolerance to variable temperatures and magnesium concentrations.|Specific amplification in multiplex PCR.|Increased specificity of primer annealing.|
The HotStarTaq procedure is fast and easy for maximum convenience.|A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.|Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.|A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.|[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.|Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|
Performance

Each lot of HotStarTaq Master Mix Kit is subjected to a comprehensive range of quality control tests, including a stringent PCR specificity and reproducibility assay in which low-copy targets are amplified. HotStarTaq Master Mix Kit outperformed kits tested from other suppliers and ensures high specificity and superior performance in hot-start PCR (see figures "Higher specificity with different primer–template systems" and "Superior performance " and table). The innovative PCR buffer provided with the kit ensures specificity over a wide range of PCR conditions, minimizing the need for optimization (see figure "Tolerance to variable temperatures and magnesium concentrations").

The combination of high specificity and easy handling makes the HotStarTaq Master Mix Kit suitable for use with complex genomic or cDNA templates (see figure "Effect of hot start on RT-PCR performance"), multiple primer pairs (see figure "Specific amplification in multiplex PCR"), and templates isolated from difficult sources or very low-copy targets (see figure "Highly sensitive single-cell PCR"). It is also suitable for projects such as genetic screening, in which large numbers of samples are amplified.

Comparison of hot-start methods 
HotStarTaq DNA Polymerase Hot-start enzyme from Supplier AII Antibody-mediated Manual Wax barrier
Specific amplification ++ + + +/– +/–
Minimal PCR optimization ++ +/– +/– – –
Easy to use ++ ++ + – –
HotStarTaq DNA Polymerase specifications

Concentration: 5 units/µl
Recombinant enzyme: Yes
Substrate analogs: dNTP, ddNTP, dUTP, biotin-11-dUTP, DIG-11-dUTP, fluorescent-dNTP/ddNTP
Extension rate: 2–4 kb/min at 72°C
Half-life: 10 min at 97°C ; 60 min at 94°C
Amplification efficiency: ≥105 fold
5'–>3' exonuclease activity: Yes
Extra A addition: Yes
3'–>5' exonuclease activity: No
Contaminating nucleases: No
Contaminating RNases: No
Contaminating proteases: No
Self-priming activity: No 

Principle

HotStarTaq Master Mix is a ready-to-use mixture of HotStarTaq DNA Polymerase, QIAGEN PCR Buffer, and dNTPs. HotStarTaq DNA Polymerase, a modified form of Taq DNA Polymerase, provides high specificity in hot-start PCR.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase is supplied in an inactive state and has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle (see figures "Superior performance in hot-start PCR" and "Higher specificity with different primer–template systems"). HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C, which can be incorporated into any existing thermal-cycler program.

QIAGEN PCR Buffer

QIAGEN PCR Buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required (see figures "Wide annealing temperature window" and "Tolerance to variable magnesium concentration").

Procedure
HotStarTaq Master Mix Kit is supplied in a convenient master mix format for maximum ease of use. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. Room-temperature reaction setup using the master mix is fast and easy — simply pipet 25 µl HotStarTaq Master Mix into each PCR tube and add 25 µl of primers and template DNA diluted in the RNase-free water provided with the kit (see figure "HotStarTaq procedure"). Pipetting steps are minimized, reducing the possibility of errors and contamination, and ensuring increased throughput and reproducibility. The kit includes a streamlined, optimized protocol for fast and easy PCR setup.
Applications

HotStarTaq Master Mix Kit is highly suitable for a wide variety of applications, including challenging applications such as amplification of: 

  • Complex genomic templates
  • Complex cDNA templates (e.g., RT-PCR)
  • Very low-copy targets (e.g., single-cell PCR)
  • Reactions with multiple primer pairs
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Feature
Specifications
Applications PCR, RT-PCR, Complex genomic templates, very low-copy targets
Enzyme activity 5'-> 3' exonuclease activity
Mastermix Yes
Reaction type PCR amplification
Real-time or endpoint Endpoint
Sample/target type Genomic DNA and cDNA
Single or multiplex Single
With/without hotstart With hotstart

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Kit Handbooks
1
HotStarTaq PCR Handbook - (EN)
HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization  
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User-Developed Protocols
1
Isolation of bacterial DNA from soil using the QIAamp® DNA Stool Mini Kit and QIAamp DNA Blood Midi Kit - (EN)
As starting material, 5 g soil was mixed with different amounts of Bacillus subtilis cells. Sensitivity was 5 x 103 cells/5g soil.
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References
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